Simplification of complex peptide mixtures for proteomic analysis: reversible biotinylation of cysteinyl peptides

A rapid means of identifying many components in an enriched mixture of proteins is enzymatic digestion of the entire protein fraction. This complex peptide mixture is then subjected to reversed-phase high performance liquid chromatography (HPLC) coupled on-line with a mass spectrometer capable of data-dependent ion selection for fragmentation (LC-tandem mass spectrometry; MS/MS). Thus, as many peptides as possible in the sample are fragmented to produce MS/MS spectra, which can then be searched against sequence databases. Ideally, one peptide from each protein in the mixture would be fragmented and identified. To this end, we employed an affinity selection method to capture cysteinyl peptides and thereby simplify the mixture. Both the captured cysteinyl and the noncysteinyl peptides are analyzed by LC-MS/MS, to increase the number of proteins identified. The method was tested on a limited set of standard proteins and applied to the analysis of a protein fraction obtained from isolated mitochondria treated with atractyloside. To further increase the number of different precursor ions selected for fragmentation, dynamic exclusion and ion selection from multiple narrow mass ranges of consecutive runs were employed.     

Selective Detection of Carbohydrates and Their Peptide Conjugates by ESI-MS Using Synthetic Quaternary Ammonium Salt Derivatives of Phenylboronic Acids

We present new tags based on the derivatives of phenylboronic acid and apply them for the selective detection of sugars and peptide-sugar conjugates in mass spectrometry. We investigated the binding of phenylboronic acid and its quaternary ammonium salt (QAS) derivatives to carbohydrates and peptide-derived Amadori products by HR-MS and MS/MS experiments. The formation of complexes between sugar or sugar-peptide conjugates and synthetic tags was confirmed on the basis of the unique isotopic distribution resulting from the presence of boron atom. Moreover, incorporation of a quaternary ammonium salt dramatically improved the efficiency of ionization in mass spectrometry. It was found that the formation of a complex with phenylboronic acid stabilizes the sugar moiety in glycated peptides, resulting in simplification of the fragmentation pattern of peptide-derived Amadori products. The obtained results suggest that derivatization of phenylboronic acid as QAS is a promising method for sensitive ESI-MS detection of carbohydrates and their conjugates formed by non-enzymatic glycation or glycosylation.

Phenylboronic acid modified solid‐phase extraction column: Preparation,characterization, and application to the analysis of amino acids in sepia capsule by removing the maltose

Maltose, a common auxiliary material of pharmaceutical preparation, may disturb the analysis of total amino acids in sepia capsule by aldolization. Therefore, it is necessary to remove the maltose through a convenient method. In this work, a phenylboronic acid modified solid‐phase extraction column has been synthesized and used to remove the maltose. The materials were synthesized by one step “thiol‐ene” reaction and the parameters of the column such as absorption capacity, recovery, and absorption specificity have been investigated. The results showed the column (0.5 cm of length × 0.5 cm of inner diameter) can absorb 4.6 mg maltose with a linear absorption and absorption specificity. Then this technique was applied in the quantification of amino acids in sepia capsule. After the optimization of the method, four kinds of amino acids, which were the most abundant, were quantified by high‐performance liquid chromatography with diode array detection. The amounts of the four kinds of amino acids are 1.5～2 times more than that without the treatment of solid‐phase extraction column, which almost overcomes the influence of the maltose. All the results indicate that the phenylboronic acid modified solid‐phase extraction column can successfully help to accurately quantify the total amino acids in sepia capsule.     

New route to synthesis of PVP‐stabilized palladium(0) nanoclusters and their enhanced catalytic activity in Heck and Suzuki cross‐coupling reactions

Herein we report a new method for the synthesis and characterization of PVP‐stabilized palladium(0) nanoclusters and their enhanced catalytic activity in Suzuki coupling and Heck reactions of aryl bromides with phenylboronic acid and styrene, respectively, under mild conditions. The PVP‐stabilized palladium(0) nanoclusters with a particle size of 4.5 ± 1.1 nm were prepared using a new method: refluxing a mixture of potassium tetrachloropalladate(II) and PVP in methanol at 80 °C for 1 h followed by reduction with sodium borohydride. Palladium(0) nanoclusters prepared in this way were stable in solution for weeks, could be isolated as solid materials and were characterized by TEM, XPS, UV–vis, and XRD techniques. The PVP‐stabilized palladium(0) nanoclusters were active catalysts in Heck and Suzuki coupling reactions of arylbromides with styrene and phenylboronic acid affording stilbenes and biphenyls, respectively, in high yield. Recycling experiments showed that PVP‐stabilized palladium(0) nanoclusters could be used five times with essentially no loss in activity in the Heck and Suzuki coupling reactions. Copyright © 2009 John Wiley & Sons, Ltd.     

基于在线富集与原位衍生技术的植物组织中内源性油菜素甾醇的超高效液相色谱-串联质谱分析方法

油菜素甾醇是一类重要的植物激素,对植物的生长发育过程起显著调节作用。已报道的油菜素甾醇分析方法存在样品前处理过程复杂和灵敏度低等问题。本研究采用C18 PEEK管填充柱为富集柱,以4-N,N-二甲氨基苯硼酸为衍生试剂,搭建了基于双泵-双六通阀的在线管内固相微萃取-超高效液相色谱-串联质谱（UPLC-MS/MS）联用系统,对油菜素甾醇进行在线富集和原位衍生,实现植物组织中内源性油菜素甾醇的自动化分析。该系统程序化地控制了油菜素甾醇的进样、萃取、衍生、分离和检测过程,有效简化前处理过程,节省人力;在线富集步骤实现了样品溶液大体积进样,提高样品利用率;原位衍生改善了油菜素甾醇电喷雾的离子化效率,使油菜素甾醇的电喷雾质谱检出限降低至pg/mL。该系统可在300 mg鲜重植物组织中检测到内源性油菜素甾醇,已成功用于水稻、油菜中多种油菜素甾醇的定量分析。     

Suzuki cross-coupling of hexachlorobenzene promoted by the Buchwald ligands

The study of cross-coupling between hexachlorobenzene and phenylboronic acid comprised five Buchwald ligands, from which 2-dicyclohexylphosphino-2′-(dimethylamino)biphenyl (DavePHOS) provided the best conversion. When excess of phenylboronic acid was used, a mixture of isomeric tri-, tetra- and pentaphenyl-substituted derivatives in the ～10:70:20 ratio was obtained, along with minor amounts of hydrodechlorination products.

     

Recognition of sugars on surface-bound cap-shaped gold particles modified with a polymer brush

A dithiolated random copolymer with pendent phenylboronic acid residues (Cys-Poly(3-acrylamidophenylboronic acid-co-2-dimethylaminopropyl methacrylamide), Cys-Poly(APBA-co-DMAPMA)) obtained by photo-iniferter method was accumulated as a polymer brush on a cap-shaped gold particles deposited on a vacuum-evaporated gold film, and the usefulness of the polymer brush as a sensing element for glycoprotein, ovalbumin (OVA), was examined by using UV-vis spectroscopy with a help of surface plasmon resonance. A similar system was constructed with a dithiolated mannose-carrying polymer, dithiolated-poly(2-methacryloyloxyethyl-D-mannopyranoside) (DT-PMEMan), prepared by the atom transfer radical polymerization (ATRP). The brush composed of this polymer was examined as a sensing element for lectin, concanavalin A (Con A). The sensor cells modified with Cys-Poly(APBA-co-DMAPMA) and DT-PMEMan showed a concentration-dependent binding of OVA and Con A, respectively, with a comparable detection limit to those with a monolayer of polymer brush-coated gold particle deposited on a glass substrate. Using this system, it can be expected to open a new perspective to various functional polymer brushes fixed to the cap-shaped gold particle on a solid substrate.     

Solid-phase synthesis of peptide and glycopeptide thioesters through side-chain-anchoring strategies

An efficient new strategy for the synthesis of peptide and glycopeptide thioesters is described. The method relies on the side-chain immobilization of a variety of Fmoc-amino acids, protected at their C-termini, on solid supports. Once anchored, peptides were constructed using solid-phase peptide synthesis according to the Fmoc protocol. After unmasking the C-terminal carboxylate, either thiols or amino acid thioesters were coupled to afford, after cleavage, peptide and glycopeptide thioesters in high yields. Using this method a significant proportion of the proteinogenic amino acids could be incorporated as C-terminal amino acid residues, therefore providing access to a large number of potential targets that can serve as acyl donors in subsequent ligation reactions. The utility of this methodology was exemplified in the synthesis of a 28 amino acid glycopeptide thioester, which was further elaborated to an N-terminal fragment of the glycoprotein erythropoietin (EPO) by native chemical ligation.     

Determination of haloperidol and reduced haloperidol in human serum by liquid chromatography after fluorescence labeling based on the Suzuki coupling reaction

A simultaneous method for the determination of haloperidol (HP) and its metabolite, reduced haloperidol (RHP), in human serum was developed by means of high-performance liquid chromatography (HPLC) with fluorescence detection. Suzuki coupling reaction with a fluorescent arylboronic acid, 4-(4,5-diphenyl-1H-imidazol-2-yl)phenylboronic acid (DPA), was employed to convert HP and RHP into highly fluorescent compounds. HP and RHP were extracted from human serum by liquid-liquid extraction with a mixture of n-hexane and isoamyl alcohol (99:1, v/v) and subsequently labeled by reaction with DPA. Separation of DPA derivatives of HP and RHP was performed on a silica column with a mixture of acetonitrile and H(2)O (90:10, v/v) containing triethylamine and acetic acid as a mobile phase. The proposed method allowed sensitive detection of HP and RHP in human serum with a detection limit (at a signal to noise ratio of 3) of 0.22 and 0.20 ng/mL, respectively. The applicability of the method for therapeutic drug monitoring (TDM) was demonstrated by analyzing human serum samples from schizophrenic patients receiving HP.     

An algorithm for interpretation of low-energy collision-induced dissociation product ion spectra for de novo sequencing of peptides

An algorithm for interpretation of product ion spectra of peptides generated from ion trap mass spectrometry is developed for de novo amino acid sequencing of peptides for the purpose of protein identification. It is based on a multi-pass analysis of product ion data using a rigorous data extraction and sequence interpretation protocol in the initial pass. The extraction/interpretation algorithm becomes more relaxed in subsequent passes, considering more of the fragment ions, and potentially more sequence candidates. The possible peptide sequences generated by the algorithm are scored according to those sequences which best explain the fragment ion spectrum. These sequences are searched against a protein database using a BLAST search engine to find likely protein candidates. The method is also suitable for locating and determining protein modifications, and can be applied to de novo interpretation of peptide fragment ions in the tandem mass (MS/MS) spectrum produced from a mixture of two peptides having similar nominal mass, but different sequences. Using a known protein, bovine serum albumin, as an example, it is illustrated that this method is rapid and efficient for MS/MS spectral interpretation. This method combined with BLAST programs is then applied to search homologies and to generate information on post-translational modifications of an unknown protein isolated from shark cartilage that does not have a complete genome or proteome database.     

非通孔多孔氧化铝基底上原位生成Pd纳米材料的制备及应用

以非双通的多孔氧化铝(AAO)(Al基底为支撑体)为模板,基底Al作为还原剂,在AAO孔道内原位合成了Pd单质(Pd/AAO)。本文详细研究了AAO阻挡层的去除以及Pd/AAO的酸腐蚀处理后的情况。采用扫描电子显微镜观察了不同条件下形成的形貌,提出了一个近似合理的生成Pd单质的机理。评价了其对苯硼酸和对溴苯甲醛Suzuki反应的活性。摩尔分数0.06%Pd催化剂在温和的实验条件下可以高效催化Suzuki反应,产率达到99.8%。Pd/AAO固体片状结构在液相反应中直接从溶液中取出即可进行下一次反应,易于分离和回收,可使用性强。此研究为有机反应异相催化剂的设计提供了一个有效的思路,在实际应用中具有一定的指导意义。     

Phenylboronate‐diol crosslinked polymer/SWCNT hybrid gels with reversible sol–gel transition

This work demonstrates the successful incorporation of functionalized single‐walled carbon nanotubes (f‐SWCNTs) into the phenylboronate‐diol crosslinked polymer gel to create a hybrid system with reversible sol–gel transition. The phenylboronic acid‐containing and diol‐containing polymers were first separately prepared by the reversible addition–fragmentation chain transfer polymerization. Covalent functionalization of single‐walled carbon nanotubes (SWCNTs) with an azide‐derivatized, diol‐containing polymer was then accomplished by a nitrene addition reaction. Subsequently, the hybrid gels were prepared by crosslinking the mixture of f‐SWCNTs and diol‐containing polymer with the phenylboronic acid‐containing polymer. The hybrid gel has been characterized by scanning electron microscopy (SEM) and rheological analysis. The SEM measurement demonstrated a homogeneous dispersion of f‐SWCNTs within the gel matrices. Rheological experiments also demonstrated that the hybrid gel exhibited storage moduli significantly higher than those of the native gel obtained from the phenylboronic acid‐containing and diol‐containing polymers. The hybrid gel can be switched into their starting polymer (f‐SWCNTs) solutions by adjusting the pH of the system. Moreover, the hybrid gel revealed a self‐healing property that occurred autonomously without any outside intervention. By employing this dynamic character, it is possible to regenerate the used gel, and thus, it has the potential to perform in a range of dynamic or bioresponsive applications Copyright © 2013 John Wiley & Sons, Ltd.     

利用亲和选择筛选法从组合化学库中筛选新药

组合化学方法可以产生大量的新化合物供新药筛选，但传统的逐一筛选法不能满足如此数量庞大的化合物，因此，针对混合物进行筛选显得非常重要，亲和选择筛选法利用药物作用的靶蛋白与潜在药物之间的亲和活性，使目标化合物与大量无亲和活性的化合物分离，然后只对有亲和活性的化合物进行结构鉴定，从而快速地从大量混合物中找到目标化合物，本文着重讨论了亲和选择筛选的的几种形，相应技术和在组合化学库筛选中的应用，亲和选择筛选大体分为两种方式：一种是将靶蛋白固定在支持物上，有亲和活力的化合物被保留，大量非亲和化合物被洗掉，另一种是在溶液中靶蛋白与混合物库孵育，然后利用受体配基复合物与不结合的游离化合性质的判别进行区分，亲和选择筛选法也适合于化合物的种类和含量都未知的天然提取物的筛选。     

Detection of Lectins by Saccharide-gold Nanoparticle Conjugates

A general functionalization strategy was reported, which enables one to conjugate saccharide(SA) on gold nanoparticle(GNP) surface without affecting SA properties. First, disulfide phenylboronic acid(Bor) functionalized GNPs(Bor@GNPs) were synthesized by the reaction of citrate stabilized GNPs of 13 nm in diameter with the mixture of Bor and pentapeptide(Cys-Ala-Leu-Asn-Asn, CALNN). Subsequently, the SA-GNP conjugates(SA@GNPs) were prepared by coupling SA to the GNP surface via the reaction of phenylboronic acid(PBA) with the cis-diol configuration in SA. The interactions of three SA@GNPs with three lectins have been analyzed by UV-visible spectroscopic and transmission electronic microscopic(TEM) techniques, respectively. The experimental results demonstrate that SA@GNPs can efficiently bind to lectins and show a great promise as optical probes for monitoring specific affinities of lectins for SA, and detecting lectins with high sensitivity.     

Activity of palladium nanoparticles on graphene oxide in the Suzuki—Miyaura reaction

A convenient express method for obtaining palladium nanoparticles on the graphene oxide support was developed. The data of transmission electron microscopy and X-ray diffraction analysis indicated the formation of palladium nanoparticles with an average size of 2 nm. The obtained nanocomposite material showed high catalytic activity in the cross-coupling reaction of bromobenzene with phenylboronic acid. The efficiency of the catalyst increases when using a mixture of organic solvents with water.     

A general method for designing combinatorial peptide libraries decodable by amino acid analysis

Herein we describe an algorithm for designing combinatorial peptide libraries for split-and-mix synthesis on solid support that are decodable by amino acid analysis (AAA) of the beads. AAA is a standard service analysis available in most biochemical laboratories, and it allows one to control the quality of the peptide on each bead, an important feature that is missing from most library decoding protocols. In the algorithm, each AA is assigned to two variable positions in the sequence grouped in a "unique pair". This arrangement limits sequence design because both the number of unique pairs U (setting the maximum number of variable AA) and the maximum number S of different AA per variable position depend on the peptide length N (U=N(N-1)/2), S=N-1). The method is therefore only suitable for focused libraries. An application example is shown for the selection of peptides with N-terminal proline or hydroxyproline catalyzing an aldol reaction from a combinatorial library of 65536 octapeptides. A simple enumeration program is available to help design combinatorial libraries decodable by amino acid analysis. The method applies to linear and cyclic peptides, can be used for nonnatural building blocks, including beta-amino acids, and should help to explore the vast chemistry of linear and cyclic peptide for catalysis and bioactivity.     

Modification of citrulline residues with 2,3-butanedione facilitates their detection by liquid chromatography/mass spectrometry

Citrullination is a post-translational modification (PTM) that results from the deimination of the amino acid arginine into citrulline by Peptidyl Arginine Deiminase enzymes and occurs in a wide range of proteins in health and disease. This modification causes a 1 Da mass shift, which can be used to identify citrullination sites in proteins by the use of mass spectrometry. However, other PTMs, such as deamidation from asparagine to aspartic acid or from glutamine to glutamic acid, can also cause a 1 Da mass shift, making correct interpretation of the data more difficult. We developed a chemical tagging strategy which, combined with an open source search application, allowed us to selectively pinpoint citrullinated peptides in a complex mixture after liquid chromatography/mass spectrometry (LC/MS) analysis. After incubation of a peptide mixture with 2,3 butanedione, citrulline residues were covalently modified which resulted in a 50 Da shift in singly charged mass. By comparison of the peptide mass fingerprint from a modified and an unmodified version of the same sample, our in-house search application was able to identify the citrullinated peptides in the mixture. This strategy was optimized on synthetic peptides and validated on a digest of in vitro citrullinated fibrinogen, where different proteolytic enzymes were used to augment the protein coverage. This new method results in easy detection of citrullinated residues, without the need for complex mass spectrometry equipment.     

A novel hybrid mode of sample injection to enhance CZE sensitivity for simultaneous determination of a pyridine-triphenylborane anti-fouling agent and its degradation products

We developed a novel hybrid sample injection mode (HSIM) that presents the combination of electrokinetic injection and vacuum injection to enhance detection sensitivity in CZE. Samples were introduced using both vacuum and electrokinetic injections simultaneously, with a water plug injected into the capillary prior to sample introduction (i.e. similarly to field-amplified sample injection, FASI). Using a sample mixture containing an anti-fouling agent applied to ship hulls, pyridine-triphenylborane and its degradation products (diphenylborinic acid, phenylboronic acid, and phenol) dissolved in ACN, the length of water plug, time, and voltage for sample introduction were optimized. The signal intensity (peak height) was found to be up to a 30-fold increased using HSIM by applying 4 kV for 4 s at the inlet end of the capillary as the cathode with supplementary vacuum in comparison with only vacuum injection for 4 s. The LODs (at a S/N of 3) for pyridine-triphenylborane, diphenylborinic acid, phenylboronic acid, and phenol were 0.88, 1.0, 21, and 23 μg/L, respectively. At the level of 0.04 mg/L, the RSDs (n=4, intra-day) for the above analytes were in the ranges of 1.9-11, 4.3-9.2, and 0.34-0.66% for peak area, peak height, and migration time, respectively. The HSIM is a simple and promising procedure useful for enhancing the sensitivity for both low-and high-mobility ions in CZE.     

Exploiting Boron Coordination: B←N Bond Supports a [2+2] Photodimerization in the Solid State and Generation of a Diboron Bis‐Tweezer for Benzene/Thiophene Separation

B←N coordination supports a [2+2] photodimerization in the solid state. The bond is defined by an orthogonal interaction between stilbazole and a phenylboronic ester to enable a stereocontrolled and rapid photoreaction. The cyclobutane photoproduct affords a novel diboron bis‐tweezer adduct that is used to separate a mixture of benzene and thiophene upon crystallization.